The blood-brain barrier serves to separate the molecular, ionic and cellular environment of the blood from that of the brain. To a major degree, this separation is achieved by inter-endothelial tight junctions of high electrical resistance which greatly diminish paracellular flux. It is clear that the permeability of the tight junctions of the blood-brain barrier is not immutable. Rather, permeability appears to undergo dynamic regulation, especially by second messenger pathways.
Acquiring the ability to manipulate the permeability of the tight junctions of the blood-brain barrier is important for a number of reasons, among which are the following:
(i) To decrease brain oedema following stroke by closing the tight junctions of the blood-brain barrier; PA1 (ii) To deliver blood-borne, membrane-impermeant drugs to the brain by reversibly opening the tight junctions of the blood-brain barrier; and PA1 (iii) To block the entry into the brain of both leukocytes that mediate an immune response, such as occurs in multiple sclerosis, and metastatic cancer cells that may form tumours. (It is believed that during cell trafficking across the endothelium, the migrating cell passes through the tight junction must therefore trigger intra-endothelial mechanisms that influence junctional permeability.)
It is also desirable to manipulate the permeability of other physiological barriers involving tight junctions.
At the molecular level, some of the components of the tight junction complex have been identified. These include ZO-1 (Stevenson et al., 1986; Willot et al., 1993), .alpha..sup.+ and .alpha..sup.- isoforms (Willot et al., Balda and Anderson, 1993), ZO-2 (Gumbiner et al., 1991), cingulin (Citi et al., 1988) and the 7H6 antigen (Zhong et al., 1993), all of which are associated with the cytoplasmic surface of the tight junction in a manner yet to be fully determined. Also, a 130 kDa phosphorprotein that appears to associate with ZO-1 and ZO-2 in an unspecified manner has recently been described (Balda et al., 1993). Finally, an integral membrane protein, termed occludin, that localizes at tight junctions has also been identified (Furuse et al., 1993).
It is clear that in endothelia and certain epithelia, the ability of tight junctions to restrict paracellular flux is not immutable. Rather, this gate function of tight junctions is capable of dynamic regulation (Madara, 1988; Rubin, 1992). In particular, the activation of signal transduction pathways either by receptor ligands or specific membrane-permeant modulators can have striking effects on the permeability of the paracellular pathway. For example, protein kinase C activation causes a substantial increase in the permeability of tight junctions in MDCK cells (Ojakian, 1981), an epithelial cell line. Cyclic AMP elevation decreases permeability in brain endothelial cells in culture, a model system for the study of the blood-brain barrier (Rubin et al., 1991). Cyclic AMP also decreases tight junction permeability in peripheral endothelial cells (Stelzner et al., 1989; Langeier et al., 1991).
The permeability properties of the tight junction also depend upon the integrity of the adherens junction. Disruption of the adherens junction by removal of extracellular Ca.sup.2+ leads to an opening of tight junctions in MDCK cells (see Martinez-Palomo et al., 1980; Gumbiner and Simons, 1986) and in endothelial cells (Rutten et al., 1987). Protein kinases appear to be involved in this indirect modulation of tight junctional integrity in MDCK cells (Citi, 1992). The Ca.sup.2+ -sensitive components of the adherens junction complex are the cadherins (reviewed by Geiger and Avalon, 1992). These transmembrane proteins mediate intercellular adhesiveness in a Ca.sup.2+ -dependent, homophilic manner via their extracellular domains. The cytoplasmic domain of the cadherins associates with three further proteins termed .alpha.-, .beta.- and .gamma.-catenin (Ozawa et al., 1989), which link the cadherins to the actin cytoskeleton and are required for cadherin adhesiveness (Hirano et al., 1987; Nagaruchi and Takeichi, 1988; Ozawa et al., 1990; Kintner, 1992; see Stappert and Kemler, 1993).
Recently, it was reported that treatment of cells with pervanadate, a tyrosine phosphatase inhibitor, or overexpression of the tyrosine kinase pp60.sup.v-src, resulted in the tyrosine phosphorylation of components of the cadherin/catenin complex (Matsuyoshi et al., 1992; Behrens et al., 1993; Hamaguchi et al., 1993). Such increased phosphorylation with associated with decreased cadherin-dependent cell adhesiveness (Matsuyoshi et al., 1992; Behrens et al., 1993; Hamaguchi et al., 1993). With respect to tight junctions, the relationship between protein tyrosine phosphorylation and structure has been studied, but, so far, with negative results. Warren and Nelson (1987) found that transfection of MDCK cells with low-levels of pp60.sup.v-src caused disruption of the adherens junction but, as revealed by electron microscopy, apparently did not affect the tight junction. Volberg et al. (1992) showed that pervanadate elicited tyrosine phosphorylation of proteins at the adherens junction in MDCK cells, but, in contrast to the findings of the present inventors, also had no effect on the tight junction, as determined by immunocytochemical examination of ZO-1 distribution.
Experiments were also carried out to investigate the effect of tyrosine phosphorylation on tight junctional permeability using bovine brain microvessel endothelial cells (BBECs) as a tissue culture model of the blood brain barrier (Rubin et al, J. Cell Biol. 115 1725-1735 (1991)). Since it is clear that the permeability of the tight junctions of epithelial cells is also subject to regulation, studies leading up to the present invention also used Madin-Darby canine kidney (MDCK) strain I cells, an epithelial cell line that possesses tight junctions of high intrinsic electrical resistance (several thousand .OMEGA.-cm.sup.2). In both cases, the cells were grown on filters so that the permeability of the tight junctions could be determined by measuring transcellular electrical resistance (TER). When cells are grown in this manner, the movement of molecules and cells across the monolayer can also be studied.
Other investigators have examined the relationship between protein tyrosine phosphorylation and tight junctional permeability, but with negative results. Warren and Nelson Mol. Cell. Biol. 7 1326-1337 (1987) found that transfection of MDCK cells with low-levels of the tyrosine kinase pp60.sup.v-src caused disruption of the adherens junction but apparently did not affect the tight junction, as revealed by electron microscopy. Using MDCK cells, Volberg et al, EMBO J. 11 1733-1742 (1992) showed that pervanadate, a tyrosine phosphatase inhibitor, elicited tyrosine phosphorylation of proteins at the adherens junction, but had no effect on the tight junction as determined by immunostaining of the tight junction associated protein, ZO-1 (see review on tight junction by S. Citi, J. Cell Biol. 121 485-489 (1993)).